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Abstract

Background

Disorders in wound healing (DWH) are common in trauma patients, the reasons being
not completely understood. Inadequate nutritional status may favor DWH, partly by
means of oxidative stress. Reliable data, however, are lacking. This study should
investigate the status of extracellular micronutrients in patients with DWH within
routine setting.

Methods

Within a cross-sectional study, the plasma/serum status of several micronutrients
(retinol, ascorbic acid, 25-hydroxycholecalciferol, α-tocopherol, β-carotene, selenium,
and zinc) were determined in 44 trauma patients with DWH in addition to selected proteins
(albumin, prealbumin, and C-reactive protein; CRP) and markers of pro-/antioxidant
balance (antioxidant capacity, peroxides, and malondialdehyde). Values were compared
to reference values to calculate the prevalence for biochemical deficiency. Correlations
between CRP, albumin and prealbumin, and selected micronutrients were analyzed by
Pearson’s test. Statistical significance was set at P < 0.05.

Keywords:

Background

Disorders in wound healing (DWH) are frequently observed post-surgically in patients
with vascular diseases and soft tissue trauma [1]. DWH are associated with a prolonged hospital stay and essentially contribute to
high morbidity and mortality [2]. Thus, apart from the individual burden, DWH generate enormous costs in the health
care system.

The pathophysiological mechanisms leading to DWH are not completely understood. Recent
studies in patients with pressure ulcers [3] support the hypothesis that general protein/energy malnutrition can considerably
increase the risk for DWH by several mechanisms. The lack of energy and nitrogen containing
metabolites, such as amino acids, may hamper wound healing (WH) by diminishing the
body’s capacity for cell repair. Low food/energy intake is often related to an insufficient
provision with essential micronutrients. It is well-known that several vitamins and
trace elements, such as retinol, ascorbic acid, 25-hydroxycholecalciferol (25(OH)D3), and zinc, are involved in collagen synthesis, cell division and epithelialization
[4-7]. Thus, nutrient intake below the actual recommendations published for healthy subjects
may increase the risk to develop DWH or even aggravate already existing DWH. Moreover,
it is still under debate whether the metabolic needs for micronutrients are considerably
higher in trauma compared with physiological conditions [3]. An insufficient intake of micronutrients may lead to intra-/extracellular deficiencies
resulting in an imbalance between pro-/and antioxidants which exerts cytotoxic effects
and, consequently, may impair WH as shown in a small patient group for selenium [8]. However, representative cross-sectional studies focusing on the assessment and evaluation
of general and specific nutritional status in a variety of patients with DWH are scarce.

Therefore, the aim of this cross-sectional study was to assess general nutritional
status, micronutrient profile and the concentration of selected biomarkers of pro-/antioxidative
balance in trauma patients with DWH in a routine clinical setting.

Methods

Patients

Following a mono-center cross-sectional design, adult trauma patients with DWH (defined
as failure to heal, i.e. wound not closed or persisting secretion within ten days
after trauma or surgery) were consecutively recruited between May and December 2006
at the Department of Orthopedics and Trauma Surgery, University of Bonn. Exclusion
criteria were defined as follows: parenteral and enteral nutrition, exclusive implant
removal, pressure ulcers as primary diagnosis, HIV infection, chronic inflammatory
bowel diseases, liver diseases, drug abuse, known pregnancy, lactation, stay in the
intensive care unit and sepsis. After enrolment, data on main diagnosis, comorbidities
and medication were obtained and the individual injury severity score [9] was determined. The time between trauma/surgery and enrolment was documented. All
patients received hospital food provided by an external caterer. Patients were asked
whether they supplemented vitamins, zinc and/or selenium.

All patients provided written, informed consent prior to enrolment. The study was
conducted according to the Declaration of Helsinki, 2004, and was authorized by the
Ethics Committee of Bonn University (No. 029/06).

Blood sampling

On the day after enrolment, blood samples were collected after an overnight fast in
EDTA or lithium coated tubes and in tubes without anticoagulant. Plasma and serum
were obtained within two hours by centrifugation at 2000 × g, 4°C for 10 min. EDTA
plasma for analysis of ascorbic acid and heparinized plasma for analysis of malondialdehyde
(MDA) were prepared as described earlier [10,11]. All samples were stored at -80°C until analysis. Laboratory parameters, except for
those investigated routinely, were analyzed in duplicate.

Anthropometric data and nutritional status

Body mass index (BMI, kg/m2) was determined by weighing the patients and asking for their height as the height
could not be determined in each patient. The BMI was evaluated according to the WHO
criteria (underweight: <18.5; normal weight: 18.5 – 24.9; overweight: 25.0 – 29.9;
obesity: ≥ 30.0) [12]. Calf and upper arm circumferences (cm) were measured in duplicate. Reference values
for calf and upper arm circumferences (sex- and age-dependent) were taken from the
NHANES study [13]; patients with values below the 10th percentile were categorized as malnourished (Table 1). General nutritional status and disease-associated weight loss were determined by
Subjective Global Assessment (SGA) [14]. Patients were classified as well-nourished (SGA A), moderately malnourished or suspected
to be malnourished (SGA B) or as severely malnourished (SGA C) (Table 2). In all cases, anthropometric data and general nutrition status were determined
by the same investigator (SCB).

Markers of pro-/antioxidant balance

The trolox equivalent antioxidant capacity (TEAC) assay [21] was used to determine antioxidant capacity (CV 1.2%) in EDTA-plasma. MDA was measured
in plasma by photometry (CV 7.4%) [22]. An ELISA kit was used to determine the concentration of peroxides (Dr. Franz Tatzber
KEG, Bisamberg, Austria; CV 4.3% according to the manufacturer) in EDTA-plasma. Uric
acid was analyzed in serum by photometry (Siemens Healthcare Diagnostics). The plasma
of several groups of staff members was analyzed to obtain values for TEAC (n = 89), peroxides (n = 21), and MDA (n = 33) from healthy controls as reference values of these parameters are still lacking.
For uric acid, the reference range was obtained from the Department of Clinical Chemistry
and Clinical Pharmacology, University Hospital Bonn.

Evaluation strategy and statistics

Data on TEAC, peroxides, and MDA were checked for normality by Kolmogorov-Smirnov
test. If normality was not reached, even after log-transformation, comparison between
patients and healthy subjects was done by Mann–Whitney U test. Correlations between CRP, albumin and prealbumin and selected micronutrients
were analyzed by Pearson’s test. Statistical significance was assumed for P < 0.05. PASW software, version 17.0 (SPSS Inc., Munich, Germany), was used for statistical
evaluation. Results are presented as means and standard deviations and as median and
quartiles, respectively.

Results

Patients

Forty-four trauma patients with DWH based on the predefined clinical criteria were
included in this study. For demographic and clinical data, see Table 4. Most patients (68%) had a soft tissue trauma and/or infections. About one third
suffered from vascular diseases or diabetes mellitus. Twelve patients were smokers.
Only a minority (n = 4) used nutritional supplements by own decision. Infections of osteosynthesis material
occurred in five and infections with methicillin-resistant Staphylococcus aureus in three patients. Two patients suffered from osteomyelitis. Median period between
trauma and study entry was 26 days (interquartile range: 15 – 68 days).

Clinical chemistry

Body composition and nutritional status

As shown in Table 1, mean BMI was 25.5 ± 5.3 kg/m2. Eleven percent of the patients were underweight, 37% were normal weight, 31% were
overweight and 21% were obese. Twenty-four percent of the patients had a calf circumference
below the 10th percentile, while an upper arm circumference below the 10th percentile
was identified in 19% of the patients. General malnutrition (SGA B/C) was prevalent
in 55% of the patients. As shown in Table 2, 15 patients (34%) were at risk for malnutrition or suspected to be malnourished
(SGA B) and nine patients (21%) were judged as severely malnourished (SGA C). Significant
weight loss (> 10% of weight within six months) was observed in 18% of the patients
with DWH.

Results on plasma protein and micronutrient concentrations are shown in Table 3. The concentrations of ascorbic acid, 25(OH)D3, β-carotene, and selenium were relatively low, and in most patients below the reference
range (Figure 1). A deficiency was also observed for albumin and, partly, for prealbumin. The prevalence
of low plasma values of pro-/vitamins, selenium, zinc as well as albumin and prealbumin
did not differ between generally well-nourished (SGA A) and malnourished (SGA B/C)
patients (data not shown).

Discussion

To the best of our knowledge, this is the first cross-sectional study collecting various
anthropometric, biochemical and clinical data to evaluate nutritional status and body
composition in a large variety of post-surgical patients with DWH. The mono-center
study was performed within clinical routine ensuring that all patients received comparable
medical therapy, care and dietetic measures. Thus, the evaluation of selected clinical
and biochemical markers may provide indication for the failure of WH.

Despite the fact that most patients were normal weight, overweight or obese and had
calf and upper arm circumferences within the reference range (Table 1), general malnutrition according to SGA was prevalent in most patients (Table 2). Parameters reflecting long-term (albumin) and short-term (prealbumin) protein supply
(Table 3, Figure 1) were in the lower normal range. Hence, general malnutrition which has been proposed
to favor DWH [3] may have contributed to the development of DWH.

Mean plasma concentrations of ascorbic acid, 25(OH)D3, β-carotene and selenium (Table 3) were below the reference values and 59 - 86% of the patients had a deficiency in
these micronutrients (Figure 1). Since post-traumatic and post-surgical metabolic events, such as inflammation [23,24] and oxidative stress [25], may generally contribute to lower plasma levels of ascorbic acid and β-carotene,
low plasma status of these micronutrients in trauma patients with DWH may be a concomitant
phenomenon considering increased levels of CRP, MDA, and peroxides. While it may not
be the reason for the development of DWH, it nevertheless should be considered that
the plasma level of micronutrients does not necessarily reflect the micronutrient
status in the wounded tissue with respect to substrate fluxes to the wound area. Such
fluxes have been observed in rats for free amino acids during early wound healing
period, leading to a relative lack of arginine in whole body [26]. Thirty percent of the patients exhibited an insufficient serum zinc status (Figure 1). Comparably low concentrations of zinc transporters, such as albumin and prealbumin,
(Table 3) may at least partly explain this observation. Another line of reasoning is the acute
phase response (APR) itself [27,28]. Elevated concentrations of acute phase proteins, such as CRP and interleukin-6,
are known to increase the expression of the zinc importer Zip14 [29] which leads to a fast zinc redistribution to organs [30]. Zorilla et al. showed that the serum zinc level is predictive for WH [5,31]. They determined the serum zinc level in preoperative patients before elective hip
replacement which may explain the different results obtained in our study (post-surgical
analysis). In contrast to other micronutrients, such as retinol, ascorbic acid, and
zinc, selenium does not have a direct physiological function in WH. However, as a
cofactor of glutathione peroxidase, selenium may reduce oxidative stress in patients
with DWH [11]. Thus, the high prevalence of selenium concentrations below the reference range in
patients with DWH (Figure 1) may contribute to DWH [11]. Insufficient serum 25(OH)D3 concentrations were observed in most of our patients (Figure 1). Since Miller et al. [32] observed an inverse association between serum vitamin D levels and inflammatory response
following hip fracture, low 25(OH)D3 concentrations in our patients may result from APR-mediated inflammation. Moreover,
immobilization of the patients may contribute to the low vitamin D status due to the
lack of UV-induced endogenous synthesis.

Disturbances in the redox state are discussed to be risk factors for delayed WH [8]. Since direct measurements of the short-lived reactive oxygen species require laborious
and expensive techniques, such as electron paramagnetic resonance, indirect methods
are used in routine clinical setting to detect an imbalance between pro- and antioxidants
[33]. This includes analysis of peroxidation products, such as MDA and peroxides, analysis
of single antioxidants (e.g., ascorbic acid, α-tocopherol, β-carotene) [34] and analysis of total antioxidant capacity reflecting the synergistic action between
endogenous (albumin, uric acid) and nutritive (ascorbic acid, α-tocopherol and β-carotene)
antioxidants [35]. Oxidative stress in patients with DWH was indicated by increased concentrations
of peroxides compared to healthy adults. TEAC was lower in patients with DWH than
in healthy controls (Table 5), probably due to lower concentrations of several endogenous (e.g., albumin) and
exogenous antioxidants like ascorbic acid (Table 3) with a high contribution to plasma antioxidant capacity. Despite the relatively
low specificity of MDA, [36], the increased values of MDA suggest an increased lipid peroxidation in patients
with DWH, which is in line with increased peroxides and reduced TEAC.

The strength of this study is the broad variety of biochemical, anthropometric, and
clinical parameters which provides a detailed picture on the micronutrient status
of trauma patients with DWH. Unfortunately, data on the quantity and quality of the
diet considering energy, protein, and micronutrients of interest and data on the kind
and dose of supplemented micronutrients were not collected. Hence, the impact of the
nutritional intake on the nutritional status of our patients cannot be assessed. The
most serious limitation of our study refers to the study design as a cross-sectional
study which does not allow any conclusion whether DWH originated from nutritional
deficiency or not. A further limitation refers to the high prevalence of vascular
diseases, diabetes mellitus and wound infections (Table 4), which are known risk factors for DWH [1]. Hence, effects on WH from these classic risk factors cannot be ruled out. Due to
these limitations, it is difficult to judge the role of selected micronutrients for
WH in vivo. In future studies with a prospective design, participants should be comparable with
regard to classic risk factors for DWH (e.g., diabetes mellitus). Also, more attention
should be paid to time-dependent changes following trauma and/or surgery.

Conclusions

Trauma patients with DWH frequently suffer from protein malnutrition and a biochemical
deficiency in several micronutrients, probably due to inflammation, increased requirement
and oxidative burden. Thus, tailored nutritional measures (fresh fruit, vegetables
and high quality protein) and/or early supplementation with selected micronutrients
are strongly recommended to hospitalized trauma patients.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SCB, SE, PS, and HG contributed to the conception and the design of the study. SCB
was responsible for data acquisition and performed statistical analysis. HG recruited
the patients. RHT was responsible for the analysis of malondialdehyde and BSW for
the analysis of biomarkers determined within clinical chemistry. CB was the clinical
advisor. SCB, SE, PS, and HG interpreted the data and drafted the manuscript. All
authors read and approved the final manuscript.

Acknowledgments

We thank Biosyn, Fellbach, Germany, for selenium analysis. We are grateful to all
patients for participation and to all physicians and nurses and technical staff for
collaboration in this study.